bs_bs_banner Biological Journal of the Linnean Society, 2013, 108, 677–687. With 4 figures The Azores bullfinch (Pyrrhula murina) has the same unusual and size-variable sperm morphology as the Eurasian bullfinch (Pyrrhula pyrrhula) JAN T. LIFJELD1*, ANTJE HOENEN2, LARS ERIK JOHANNESSEN1, TERJE LASKEMOEN1, RICARDO J. LOPES3, PEDRO RODRIGUES3,4 and MELISSAH ROWE1 1Natural History Museum, University of Oslo, PO Box 1172 Blindern, 0318 Oslo, Norway 2Electron Microscopical Unit for Biological Sciences, Department of Molecular Biosciences, University of Oslo, PO Box 1041 Blindern, 0316 Oslo, Norway 3CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado, Universidade do Porto, 4485-661 Vairão, Portugal 4CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado, Polo dos Açores, Universidade dos Açores, 9501-801 Ponta Delgada, Portugal Received 25 July 2012; revised 25 September 2012; accepted for publication 25 September 2012 The Azores bullfinch is endemic to the island of São Miguel in the Azores archipelago and the sister species to the Eurasian bullfinch. Here we show that the spermatozoa of the two species have similar ultrastructure and gross morphology. Thus, the unusual and supposedly neotenous sperm morphology previously described for the Eurasian bullfinch appears to be an ancestral trait that evolved before the two taxa diverged. In addition, the coefficients of variation in total sperm length, both within and among males, were high in both species and exceed any previously published values for free-living passerines. Such high sperm-size variation is typically found in species with relaxed sperm competition. However, the high variance in mean sperm length among Azores bullfinches is surprising, because the trait has high heritability and this small, insular population shows clear signs of reduced genetic diversity at neutral loci. A possible explanation for this apparent contradiction is that the Azores bullfinch has retained more diversity at functional and fitness-related loci than at more neutral parts of the genome. Finally, we also present data on relative testis size and sperm swimming speed for the Eurasian bullfinch, and discuss the hypothesis that the small and putatively neotenous sperm in bullfinches has evolved in response to lack of sperm competition. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 677–687. ADDITIONAL KEYWORDS: Fringillidae – Passeriformes – sperm competition – sperm length – sperm velocity – testis size. INTRODUCTION the order Passeriformes has a unique and highly conserved sperm structure: sperm are filiform, the Sperm cells are extremely variable in shape and size head (i.e. acrosome and nucleus) is corkscrew-shaped, across the animal kingdom (Cohen, 1977; Pitnick, the midpiece is elongated and wrapped around the Hosken & Birkhead, 2009). Phylogeny explains much flagellum, and a helical membrane (microtubule of this variation, which makes sperm morphology an helix) is coiled along the length of the head and informative trait for phylogenetic inference and tax- midpiece (Koehler, 1995). This is in contrast to onomy (Jamieson, Ausió & Justine, 1995). In birds, the non-passeriform orders, in which sperm cells, while also filiform, typically have a smooth and cylin- drical head, a short midpiece, and no helical mem- *Corresponding author. E-mail: [email protected] brane (Jamieson, 2006). There are also a number © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 677–687 677 678 J. T. LIFJELD ET AL. of ultrastructural details visible through scanning (2) the Himalayan clade with the orange bullfinch (SEM) and transmission electron microscopy (TEM) (P. auranthiaca), the red-headed bullfinch (P. erythro- that make the sperm of oscine passerines (Passeri) cephala), and the grey-headed (or Beavan’s) bullfinch unique among birds, including a single, fused mito- (P. erythaca); and (3) the Southeast Asian clade with chondrion, the lack of a proximal centriole (but see the brown bullfinch (P. nipalensis) and the white- Aire & Ozegbe, 2012) and an elongated, pointed acro- cheeked bullfinch (P. leucogenis) (Töpfer et al., 2011). some (Koehler, 1995; Jamieson, 2006). Importantly, The last-named clade is more basal in the bullfinch passerine sperm vary greatly in size and dimensions phylogeny, leaving the Himalayan clade as the sister (Koehler, 1995; Jamieson, 2006; Immler & Birkhead, to the Eurasian clade. To date, sperm morphology 2007; Kleven et al., 2008): the mean total length of has not been described in these species, with the formalin-fixed sperm ranges between 40 and 280 mm exception of one individual of the grey-headed bull- among the more than 200 passerine species screened finch (see Birkhead et al., 2006). In that instance, so far in our lab (Lifjeld et al., 2010; own unpublished sperm were found to have a pointed and spiral- data). Most of this variation is attributable to the shaped head (i.e. the typical passerine head shape length of the midpiece (Immler & Birkhead, 2007), contrasting markedly with that of the Eurasian bull- which contains the fused mitochondria important for finch sperm), although sperm length was equally energy production and sperm movement (Cummins, short with an extremely small midpiece. This finding 2009). would suggest that the neotenous sperm of the Eura- One species stands out as an exception to the sian bullfinch might be an autapomorphy (i.e. con- typical passerine sperm, namely the Eurasian bull- fined to the Eurasian bullfinch only). However, more finch Pyrrhula pyrrhula (Birkhead et al., 2006, 2007). species in this genus need to be analysed before any In this species, spermatozoa are non-filiform and definitive conclusions on the evolution of the unusual have a cylindrical head with a rounded, not pointed sperm morphology in the Eurasian bullfinch can be acrosome and a very short midpiece that lacks a made. helical membrane (Birkhead et al., 2006). In addition, The Azores bullfinch is endemic to the island São ultrastructural studies reveal several primitive traits Miguel in the Azores archipelago in the Atlantic not found in other oscine passerines, including a Ocean (BirdLife International, 2010; del Hoyo et al., proximal centriole and several small mitochondria 2010). The breeding range of this species is restricted instead of one large, fused mitochondrion (Birkhead to a 100-km2 area of native laurel forest on the et al., 2007). However, the deviant structural charac- eastern side of the island, and the current population teristics of the sperm of the Eurasian bullfinch are size is estimated at about 1000–1600 individuals actually present in the spermatids (i.e. immature (Monticelli et al., 2010; Ceia et al., 2011). The species sperm) of other passerines. Thus, suppression of the has recently been downlisted from ‘Critically Endan- final stages of spermatogenesis is thought to cause gered’ to ‘Endangered’ on the IUCN Red List, as the the unusual morphology of the Eurasian bullfinch population size is considered to be stable (BirdLife sperm (Birkhead et al., 2007). It has been further International, 2010). However, the species underwent hypothesized that the neotenous sperm of this species a severe population bottleneck in the 20th century, may ultimately result from a lack of sperm competi- and the current population shows clear signs of low tion (Birkhead et al., 2006; Birkhead & Immler, 2007). genetic diversity. There seems to be only one mito- Several lines of indirect evidence, including low rela- chondrial haplotype in the current population (Töpfer tive testis mass, few sperm stored in the seminal et al., 2011; R. J. Lopes, unpubl. data). A microsatel- glomera and low sperm swimming speed (Birkhead lite analysis also revealed very low levels of hetero- et al., 2006), as well as relatively short sperm with zygosity and allelic diversity relative to continental high coefficients of intermale variation in sperm total populations of the Eurasian bullfinch (R. J. Lopes, length and size components (Calhim, Immler & Birk- unpubl. data). head, 2007), support this assumption. However, to Here we present the first description of the sperm date there are no paternity studies published on the morphology and its variation in the Azores bullfinch. species to confirm genetic monogamy. Our study has two main objectives. First, we The evolutionary origin of the unusual sperm mor- wanted to know whether the neotenous sperm mor- phology of the Eurasian bullfinch is poorly under- phology of the Eurasian bullfinch is also present in stood. According to the most recent classifications the Azores bullfinch. If so, the trait might be a (del Hoyo, Elliott & Christie, 2010; Sangster et al., synapomorphy present in the last common ancestor 2011; Gill & Donsker, 2012), the genus Pyrrhula of the entire Eurasian bullfinch clade, as opposed to consists of seven species. These species fall into three a more recent event within the Eurasian bullfinch major phylogenetic clades: (1) the Eurasian clade lineage. We studied the sperm morphology of both with P. pyrrhula and the Azores bullfinch (P. murina); species using SEM and TEM for ultrastructural © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 677–687 SPERM MORPHOLOGY IN BULLFINCHES 679 details, and high-resolution light microscopy for MATERIAL AND METHODS gross morphology. Second, we wanted to examine if the Azores bull- The analyses of sperm morphology